The Hedgehog signalling molecule (Hh) is a secreted autoproteolytic protein that activates the Hedgehog protein signalling pathway, which is a signalling pathway that plays a fundamental role in the morphogenesis of numerous tissues, in particular in the formation of the endoderm and of the embryonic axis, development of the brain and of the hair follicles, as well as in cellular proliferation, and is thought to be involved in tissue maintenance and repair in adults (Ingham et al., Genes Dev., 2001, 15, 3059-3087; Marti et al., Trends Neurosci., 2002, 25, 89-96; Weschler et al., Annu. Rev. Neurosci., 2001, 24, 385-428).
The Hedgehog protein and the associated transduction pathway, initially demonstrated in Drosophila, are conserved in vertebrates and invertebrates. A single homolog of Hh is present in Drosophila, whereas three homologs of Hh: Sonic (Shh), Indian (Ihh) and Desert (Dhh) are present in mammals. Among these three homologs, Shh has received most study owing to its extended expression profile during development. Shh participates in ventralization of the neural tube, specifying the early phenotype of several neuronal types along the ventral midline (motoneurons of the spinal cord, dopaminergic or cholinergic neurons), and inducing the generation of the oligodendrocyte precursors starting from the ventral spinal cord. Moreover, Shh induces survival of the GABAergic and dopaminergic neurons, orients the future of the serotoninergic precursors and prevents death of dopaminergic neurons caused by the toxin MPP. Finally it induces proliferation of the granule cell precursors in the early postnatal cerebellum. As for the other members of the Hedgehog family, they participate in the development of bone tissue (Ihh), of the testes and of the peripheral nerves (Dhh), respectively. Moreover, the results obtained with Shh also apply to Dhh and Ihh.
Shh is synthesized in the form of a precursor that undergoes a series of post-translational modifications during which the protein is cleaved by an enzyme activity present in its C-terminal portion. This autoproteolysis generates a C-terminal fragment (ShhC) and an N-terminal fragment (ShhN) that represents the active fragment. During this reaction, addition of a cholesterol molecule in the C-terminal portion of ShhN is also observed, which promotes anchorage of ShhN to the membrane. Finally an acetyl transferase allows addition of a palmitate molecule on a cysteine residue near the N-terminal end. These events produce a biologically active Shh protein. Secretion of the protein is dependent on the protein Dispatched (Disp), two isoforms of which, Disp 1 and 2, exist in mammals (Heretsch et al., 2010, Bioorg. Med. Chem. Lett. 18: 6613-6624).
The soluble ShhN fragment transmits its action via a complex containing two transmembrane proteins: Patched (Ptc), a protein with 12 transmembrane domains having a structure of the transporter type, and Smoothened (Smo), a protein with 7 transmembrane domains homologous to the members of the superfamily of receptors coupled to proteins G (RCPG). In mammals, there is a second form of Patched: Ptc2.
In the absence of its ligand Shh, Ptc inhibits Smo. An intracellular cascade, involving a great many factors including the protein Suppressor of Fused (SuFu) and the protein PKA, is then induced. SuFu is a negative regulator of the Shh signalling pathway, it can bind to the three transcription factors of the Gli family and regulate their activation. Moreover, deletion of Sufu results in activation of the pathway. The transcription factors of the Gli family are then phosphorylated, ubiquitinylated and then cleaved in their negative form (GliR) by the proteasome, GliR penetrates into the nucleus and transcription is inactive. When Shh binds to Ptc, the inhibition that the latter exerts on Smo is raised with nuclear translocation of the active form of the Gli transcription factors (GliA) and transcriptional activation of target genes such as ptc and gli1.
The protein Hedgehog interacting protein (Hip) is capable of binding Shh with an affinity comparable to that of the protein Ptc (Traiffort et al., J. Neurochem., 2010, 113: 576-590). Hip is regarded as a negative modulator of the pathway because by binding Shh it decreases the amount of ligand available for activating the signalling pathway via Ptc. Cdo and Boc belong to the family of cell surface proteins possessing immunoglobulin and fibronectin motifs of type III. These proteins regulate the Shh signalling pathway positively by facilitating presentation of the ligand Shh to Ptc, by increasing the amounts of morphogen in the vicinity of the target cells and possibly by affecting the activity of the Gli proteins (Heretsch et al., 2010, Bioorg. Med. Chem. Lett 18: 6613-24: Scales and de Sauvage, 2009, Trends Pharmacol. Sci. 30: 303-312).
The regulatory role of the Hedgehog protein signalling pathway during embryonic development has been studied extensively: Hh has been associated with the processes of maintenance and repair of normal tissue, with spatiotemporal regulation of proliferation and differentiation, thus allowing developing tissues to reach their correct size with the appropriate cell types and appropriate degrees of vascularization and innervation. It has notably been implicated in the development of the central nervous system (Dessaud et al., 2008, Development, 135: 2489-503). The essential role of the Hh signalling function is demonstrated by the dramatic consequences of defects in this signalling pathway in the human fetus, such as holoprosencephaly observed with mutants of Shh (Traiffort et al., J. Biol. Chem., 2004, 279: 42889-42997).
More recently the Shh pathway was identified in the adult brain, where the amino-terminal active form of the molecule is expressed in a great many regions of the mature nervous system suggesting new roles for this pathway. In fact, it notably participates in establishment and maintenance of neurogenic niches and regulates the proliferation of neural or glial precursors in the adult brain (Traiffort et al., 2010, J. Neurochem., 113: 576-590). Modulation of the Shh signalling pathway therefore represents a challenge for the development of therapies for neurodegenerative diseases. Studies have already demonstrated positive effects of activation of the Shh signalling pathway by the Shh protein itself, on reduction of behavioral disorders in rats with Parkinson's disease (Tsuboi et al., 2002, Exp. Neurol. 173: 95-104) or on remyelinization of neurons in rats with multiple sclerosis (Mastronardi et al. 2004, J. Immunol. 172: 6418-26). Moreover, it has been shown that activation of the Shh signalling pathway by a Smo agonist allows an increase in proliferation of neural precursors at the level of areas of neurogenesis in the adult mouse (Machold et al., 2003, Neuron., 39: 937-950). However, the Smo agonists remain few in number and are still poorly characterized.
Dysfunctions of the Shh signalling pathway have also been associated with many cancers. In fact, mutations that inactivate Ptc are associated with Gorlin syndrome or basal cell nevus syndrome, an autosomal dominant disease characterized by craniofacial and cerebral malformations, but especially by a high incidence of various tumors, more particularly of basal cell carcinomas at the cutaneous level and medulloblastomas, at the cerebellum level. Mutations of the human genes Ptc or Smo are also observed in primary neuroectodermal tumors of the central nervous system, principally medulloblastomas (30% of cases), but also in sporadic forms of basal cell carcinomas (40% and 20% of cases for Ptc and Smo respectively). Moreover, mutations of Shh are also associated with basal cell carcinomas. Other types of tumors have also been associated with a defect of the Hedgehog signalling pathway, and the localization of these tumors is closely correlated with the expression sites of the components of the pathway during embryonic development (Scales and de Sauvage, 2009, Trends in Pharmacol. Sci., 30: 303-312). We may mention, as nonlimiting examples: breast cancers and meningiomas associated with mutations of Ptc, glioblastomas associated with mutations of Gli, gastrointestinal cancers, notably primary cancers of the stomach and colon, cancers of the prostate and of the bladder, fibromas and dermoid cysts of the ovary, rhabdomyosarcomas, small cell lung cancers, squamous cell oral carcinomas.
Owing to the crucial role of the Hedgehog protein signalling pathway in numerous physiological and pathological processes, the components of this pathway, such as the proteins Smoothened (Smo 1 and Smo 2), Frizzled (Fz 1 to Fz 10), Patched (Ptc 1 and Ptc 2), the proteins Dispatched (Disp 1 and Disp 2) or also the protein Hip represent targets for the development of new molecules capable of modulating (activating or inhibiting) this pathway and therefore of regulating development positively or negatively [proliferation, differentiation, migration, survival (apoptosis)] and/or the activity of differentiated cells and of stem cells, in vitro and/or in vivo in the embryo or in the adult.
It has been demonstrated that such molecules are useful in the treatment of tumors associated with hyperactivation of the Hedgehog pathway (Scales and de Sauvage, 2009, Trends in Pharmacol. Sci., 30: 303-312). Such molecules might therefore be usable in the treatment of various tumors such as nerve tissue tumors (medulloblastomas, primary neuroectodermal tumors, glioblastomas, meningiomas and oligodendrogliomas), skin tumors (basal cell carcinomas, trichoepitheliomas), tumors of muscle and bone tissues (rhabdomyosarcomas, osteosarcomas, melanomas) and tumors of other tissues (kidney, bladder, prostate, lung, stomach, pancreas, breast, liver).
Such molecules are also useful in the treatment of disorders of the neurodegenerative type requiring blocking of the Hedgehog pathway (Parkinson's disease, Huntington's chorea, Alzheimer's disease, multiple sclerosis, motor neuron disease), and diseases in which blocking of the Hedgehog signalling pathway might be beneficial, such as diabetes.
Such molecules are also useful in medical or surgical treatment (plastic or reconstructive surgery, grafts of tissues or of organs) of numerous acute, subacute or chronic, genetic or acquired disorders—involving a tissue dysfunction connected with deregulation of the Hedgehog pathway—for inducing the formation, regeneration, repair and/or increase in activity of tissues such as nervous tissue [central nervous system (brain) and peripheral nervous system (sensory, motor, sympathetic neurons)], bone, cartilage, testes, liver, spleen, intestine, pancreas, kidneys, smooth and skeletal muscles, heart, lungs, skin and hair, mucosae, blood cells and cells of the immune system. As nonlimiting examples of these disorders, we may notably mention neuropathies and the associated neuromuscular diseases, diabetes, alopecia, burns, ulcerations (skin and mucosae) and disorders of spermatogenesis.
Various molecules capable of modulating the activity of the Hedgehog pathway have been identified.
Firstly, the Hedgehog proteins and derived polypeptides (fragments, variants etc.), notably agonists and antagonists of the Hedgehog proteins (International Application PCT WO 01/98344 in the name of BIOGEN); owing to their size, these proteins and the derived polypeptides cannot cross the blood-brain barrier and therefore cannot be administered systemically, notably for treating brain tumors connected with hyperactivation of the Hedgehog protein signalling pathway. Moreover, such molecules are of low stability, and are difficult to produce and purify. Conversely, molecules exist that inhibit the effect of the Shh ligand, robotnikinin and the 5E1 monoclonal antibody.
The Hh signalling pathway can also be modulated further downstream. The inhibitory effect of Ptc on Smo can be modulated, for example. It is increased by statins and reduced by oxysterols by a mechanism that is not yet properly understood (Heretsch et al., Bioorg. Med. Chem. Lett., 2010, 18: 6613-6624). Natural products (Physalin F) or synthetic products (GANT58, GANT61, HPI-1) are also known to inhibit the binding of the Gli transcription factors to the DNA in the nucleus.
However, most research is focused on the discovery of modulators acting at the level of the Smoothened receptor:                heterocyclic organic molecules inhibiting or activating (SAG and derivatives) the Hh signalling pathway: International Application PCT WO 01/74344 in the name of CURIS; Chen et al., PNAS, 2002, 99, 14071-14076,        purmorphamine, a small molecule activating the Hh signalling pathway: Wu et al., Chemistry & Biology, 2004, 1229-1238,        nitrogen-containing heterocyclic molecules: International Applications PCT WO 01/19800, WO 01/26644 and WO 02/30421 in the name of CURIS; Kamenetsky et al., J. Biol., 2002, 1, 1-19,        plant steroids derived from Veratrum spp. (jervine, cyclopamine and cycloposine) and from Solanum spp. (solanidine), substituted in position 16, 17 or 18 with an amine or an amine derivative, and cholesterol: U.S. Pat. No. 6,432,970 and International Applications PCT WO 99/52534 and WO 01/27135 in the name of JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE; U.S. Pat. No. 6,291,516; International Application PCT WO 00/41545 in the name of ONTOGENY INC.; International Application PCT WO 02/30462 in the name of CURIS; Taipale et al., Nature, 2000, 406, 1005-1009; Berman et al., Science, 2002, 297, 1559-1561. However, it was demonstrated that concentrations of cyclopamine above 10 μM proved to be cytotoxic for the cells (Borzillo et al., Curr. Top Med. Chem., 2005, 5(2), 147-157). Moreover, the effects in vivo of cyclopamine on tumor growth have been called into question as they might be connected with an activity outside of the tumor itself (Yauch et al., 2008, Nature, 455: 406-410). A derivative of cyclopamine (IPI-926) is currently in clinical phase II (Mahindroo et al., J. Med. Chem., 2009, 52, 3829; Tremblay et al., J. Med. Chem., 2009, 52: 14, 4400-4418),        mifepristone (17β-hydroxy 11β-(4-dimethylamino phenyl) 17α-(prop-1-ynyl)estra-4,9-dien-3-one), also called RU-486 or RU-38486 (French patent FR 2 850 022 in the name of the CNRS) for which an inhibitory activity on the activity of the Hedgehog protein signalling pathway has been demonstrated,        the molecules SANT74 and SANT75 having a structure similar to that of SAG, synthetic activating compound of the chlorobenzothiophene type (CAS No.: 364590-63-6) are also known to be stable inhibitors for effectively controlling the conformation of the activator Smo (Yang et al., The Journal of Biological Chemistry, published Apr. 14, 2009).        

More recently, other compounds that inhibit the Hedgehog signalling pathway have also been described (Peukert and Miller-Moslin, 2010, ChemMedChem 5: 500-512; Low and De Sauvage, 2010, J. Clin. Oncol.; Ng and Curran, 2011, Nature Review Cancer):                Inhibitors based on bisamide (International Application PCT WO 2007/059157 in the name of GENENTECH INC. and CURIS INC.) and on pyridyl (International Application PCT WO 2006/028958 in the name of GENENTECH INC. and CURIS INC; US Patent 2009/0281089 in the name of GENENTECH INC.). One of the pyridyl-based compounds, GDC-0449 (clinical phase II) has shown its efficacy in a patient with medulloblastoma metastases. However, the patient gradually developed resistance to the molecule. A mutation of an aspartic acid (D473H) of Smo appeared. This interferes with the compound's ability to bind to Smo and inhibit this pathway. A mutation of the same amino acid was identified in a mouse model with medulloblastomas, treated with this compound (Yauch et al., 2009, Science 326: 572-574).        Inhibitors developed by the company Novartis. For example, LDE225 (clinical phase II) has been tested for treating medulloblastomas in the mouse model and has induced regression of these tumors. However, resistance was observed over the course of the treatment. A study has revealed several mechanisms of resistance including chromosomal amplification of Gli2 and, more rarely, point mutations of the Smo receptor that lead to reactivation of tumor growth. Positive regulation of phosphatidylinositol 3-kinase (PI3K) signalling has also been identified (Buonamici et al., 2010, Sci. Transl. Med., 51-70).        Inhibitors developed by Bristol-Myers Squibb Inc. such as BMS-833923 (XL-139).        Inhibitors described by the company Pfizer Products, Inc. (WO 2008/075196 and US 2009/0005416). PF-04449913 is currently in clinical phase II.        Inhibitors described by the company MERCK (Dessole et al., 2009, Bioorg. & Med. Chem. Lett. 19: 4191-4195).        The compounds LEQ506 of Novartis and TAK-441 of the company Millennium have also just entered clinical phase I.        Inhibitors based on acyl-ureas, -thioureas and -guanidines have also been protected as modulators of the Hedgehog pathway (WO 2009/130422 and WO 2011/010013 in the name of the CNRS). The latter have the advantage of being easy to prepare in comparison with the other existing molecules. Moreover, the acyl-guanidine derivatives are water-soluble.        